Abstract:

In the present study our investigation shows that phosphorylation of tonoplast proteins purified from maize (Zea mays L.) roots increases obviously V-type H⁺-ATPase (V-ATPase) activities of ATP hydrolysis and H⁺ transport. Further research indicates that some of the purified tonoplast proteins can be thiophosphorylated and one band about 69 kD is identified as subunit A with antibody against subunit A of V-ATPase. In order to determine the phosphorylation site(s) in subunit A of V-ATPase, the subunit A band at 69 kD was isolated from thiophosphorylated gel and then completely digested by trypsin. After purification of these enzymatic lysis fragments with RP-HPLC, the molecular weight of phosphorylated peptide fragment was determined as 573.83 Da with mass spectrometry. Data search indicates that subunit A can generate 61 peptide fragments after tryptic digestion, of which only F56 with molecular weight of 573.66 Da is close to that of the identified fragment, and F56 can only be phosphorylated at Ser₅₂₅. Therefore our research suggests that Ser₅₂₅ is the potential phosphorylation site of V-ATPase subunit A in maize roots. To our knowledge, this is the first time to determine the phosphorylation site of V-ATPase subunit A in plants.

Key words: V-ATPase, subunit A, phosphorylation site, tonoplast, maize